Glial cells monitor and respond to neural activity by conditioning the extacellular milieu, signaling within glial cell networks as well as by sending signals back to neurons. Unlike neurons, which use electrical signals to communicate, glial cells possess a form of Ca2+ based excitability, where they generate and propagate intracellular Ca2+ signals as waves over long distances in response to monitored synaptic activity. We aim to understand the principles and mechanisms that govern intracellular calcium signals in glial cells and neurons. One objective is to understand processes that support temporal and spatial characteristics of Ca2+ signals within glial cells and between neurons and glia. A second objective is to characterize the specific signal traffic between myelinating Schwann cells and the axons they myelinate in peripheral nerves. Myelinating Schwann cells monitor and respond to impulse traffic along the axons they myelinate. In previous work, we found that Ca2+ wave propagation in glial cells was saltatory, due to the underlying regenerative Ca2+ release process. Regenerative Ca2+ release occurs at wave amplification sites, which are specialized Ca2+ release sites found 5 to 7 micrometers apart along glial cell processes. A number of proteins involved in Ca2+ signaling such as IP3Rs, SERCA, calreticulin, and RyR are expressed together at these specialized release sites in high density patches. Based on these findings we hypothesize that the wave amplification sites are indeed specialized microdomains of Ca2+ release where a number of specialized proteins involved in aspects of Ca2+ signaling are expressed together forming a complex protein machine. Some of these protein components are present in different membrane systems including the plasma membrane, the endoplasmic reticulum membrane and mitochondrial membrane. The protein components of such a cellular machine interact with each other in a coordinated fashion. In addition, we hypothesize that such sites are organized in cellular processes at strategic locations in order to serve the signaling needs of the cell. This anchoring occurs by specific interaction of signaling proteins with molecular scaffolds and adapter proteins, which anchor the entire assembly to cytoskeletal components. We are engaged in a comprehensive molecular characterization of this putative signaling microdomain using proteomic technologies. We are in the process of isolating Ca2+ signaling microdomains from oligodendrocyte progenitor cells and astrocytes from rodent brains. Oligomeric protein complexes will be separated using immunoaffinity strategies and will be analyzed using proteomic methods. Two approaches will be used. In one, mass spectra of proteolytic digests of proteins spots excised from electrophoresis gels will be analyzed by direct mass spectrometry. MALDI-TOF will be used for initial peptide mass finger printing. Masses of peptides derived from an in-gel proteolytic digestion will be measured and searched against a computer-generated list formed from the simulated digestion of a protein database using the same enzyme. In the second approach, proteins whose full-length sequences are not yet available will be analyzed using a nano-electrospray technology. Proteolytic peptide pools of unknown proteins will be separated by liquid chromatography followed by tandem electrospray mass spectrometry (LC-MS/MS). The electrospray technique generates peptide sequence tags, which enable searching in EST data bases of partially sequenced proteins. A comprehensive description of the component protein machines that comprise signaling microdomains in glial cells is expected to result from this investigative effort. The second objective is to specifically investigate the signaling between Schwann cells and the axons they myelinate. Towards this end, the Section is engaged in two separate avenues of research. In one, a comprehensive characterization of the distribution of Ca2+ signaling proteins in the nodes of Ranvier, internodes and the axoglial apparatus in the sciatic nerve is underway. Preliminary experiments show a characteristic localization of IP3Rs presumably dictated by the unique architecture of the myelin sheaths around the axons in the nodes and internodes. In a second study, a transgenic mouse line has been developed in the Section in which a Ca2+ sensitive photoprotein, cameleon YC 3.60 is expressed under the control of the promoter for the S-100b protein uniquely expressed by the Schwann cells in the peripheral nervous system and astrocytes in the central nervous system. This animal model allows us to continuously monitor physiological signaling within these glial cells associated with action potential traffic in the myelinated axons as well as neural activity dependent astrocytic signals. Efforts are underway to record such signals in cells and tissue slices isolated from these mice as well as the intact animal itself using multi-photon confocal microscopy.